Supraphysiological FOXP3 expression in human CAR-Tregs results in improved stability, efficacy, and safety of CAR-Treg products for clinical application.

The forkhead family transcription factor (FOXP3) is an essential regulator for the development of regulatory T cells (Tregs) and orchestrates both suppressive function and Treg lineage identity. Stable expression of FOXP3 enables Tregs to maintain immune homeostasis and prevent autoimmunity. However, under pro-inflammatory conditions, FOXP3 expression in Tregs can become unstable, leading to loss of suppressive function and conversion into pathogenic T effector cells. Therefore, the success of adoptive cell therapy with chimeric antigen receptor (CAR) Tregs is highly dependent on the stability of FOXP3 expression to ensure the safety of the cell product. To warrant the stable expression of FOXP3 in CAR-Treg products, we have developed an HLA-A2-specific CAR vector that co-expresses FOXP3. The transduction of isolated human Tregs with the FOXP3-CAR led to an increase in the safety and efficacy of the CAR-Treg product. In a hostile microenvironment, under pro-inflammatory and IL-2-deficient conditions, FOXP3-CAR-Tregs showed a stable expression of FOXP3 compared to Control-CAR-Tregs. Furthermore, additional exogenous expression of FOXP3 did not induce phenotypic alterations and dysfunctions such as cell exhaustion, loss of functional Treg characteristics or abnormal cytokine secretion. In a humanized mouse model, FOXP3-CAR-Tregs displayed an excellent ability to prevent allograft rejection. Furthermore, FOXP3-CAR-Tregs revealed coherent Treg niche-filling capabilities. Overexpression of FOXP3 in CAR-Tregs has thereby the potential to increase the efficacy and reliability of cellular products, promoting their clinical use in organ transplantation and autoimmune diseases.

Long-Lasting Imprint in the Soluble Inflammatory Milieu Despite Early Treatment of Acute Symptomatic Hepatitis C.

BACKGROUND: Treatment with direct-acting antivirals (DAAs) in patients with chronic hepatitis C infection leads to partial restoration of soluble inflammatory mediators (SIMs). In contrast, we hypothesized that early DAA treatment of acute hepatitis C virus (HCV) with DAAs may normalize most SIMs. METHODS: In this study, we made use of a unique cohort of acute symptomatic hepatitis C patients who cleared HCV with a 6-week course of ledipasvir/sofosbuvir. Plasma samples were used for proximity extension assay measuring 92 proteins. RESULTS: Profound SIM alterations were observed in acute HCV patients, with marked upregulation of interleukin (IL)-6 and CXCL-10, whereas certain mediators were downregulated (eg, monocyte chemoattractant protein-4, IL-7). During treatment and follow-up, the majority of SIMs decreased but not all normalized (eg, CDCP1, IL-18). Of note, SIMs that were downregulated before DAA treatment remained suppressed, whereas others that were initially unchanged declined to lower values during treatment and follow-up (eg, CD244). CONCLUSIONS: Acute hepatitis C was associated with marked changes in the soluble inflammatory milieu compared with both chronic hepatitis patients and healthy controls. Whereas early DAA treatment partly normalized this altered signature, long-lasting imprints of HCV remained.

Treg-specific IL-2 therapy can reestablish intrahepatic immune regulation in autoimmune hepatitis.

Autoimmune hepatitis (AIH) is a chronic autoimmune inflammatory disease that usually requires life-long immunosuppression. Frequent relapses after discontinuation of therapy indicate that intrahepatic immune regulation is not restored by current therapies. As steroid therapy preferentially depletes intrahepatic regulatory T cell (Tregs), immune regulation might be re-established by increasing and functionally strengthening intrahepatic Tregs. In recent clinical trials with low dose IL-2, the Treg compartment was strengthened in autoimmune diseases. Therefore, we tested complexed IL-2/anti-IL-2 to increase the selectivity for Tregs. We used our model of experimental murine AIH (emAIH) and treated the mice with complexed IL-2/anti-Il-2 in the late course of the disease. The mice showed increased intrahepatic and systemic Treg numbers after treatment and a reduction in activated, intrahepatic effector T cells (Teffs). This resulted in a reduction in liver-specific ALT levels and a molecular pattern similar to that of healthy individuals. In conclusion, complexed IL-2/anti-IL-2 restored the balance between Tregs and Teffs within the liver, thereby improving the course of emAIH. Treg-specific IL-2 augmentation offers new hope for reestablishing immune tolerance in patients with AIH.

The impact of hepatitis B surface antigen on natural killer cells in patients with chronic hepatitis B virus infection.

BACKGROUND & AIMS: During chronic hepatitis B virus (HBV) infection, suppressed functionality of natural killer (NK) cells might contribute to HBV persistence but the underlying mechanisms remain elusive. A peculiar feature of HBV is the secretion of large amount of hepatitis B surface antigen (HBsAg). However, the effect of HBsAg quantities on NK cells is unclear. The aim was to determine the effects of HBsAg quantities on NK cell functionality in patients with chronic hepatitis B (CHB). METHODS: Eighty CHB patients were included and categorized into four groups based on their HBsAg levels. As a control, 30 healthy donors were enrolled. NK cell frequency, phenotype and function were assessed using flow cytometry and correlated with HBsAg levels and liver enzymes. RESULTS: Compared to the healthy controls, a reshaping of NK cell pool towards more CD56bright NK cells was observed during CHB infection. Importantly, NK cells in patients with low HBsAg levels (<100 IU/mL) displayed an activated phenotype with increased expression of activation makers CD38, granzyme B and proliferation marker Ki-67 while presenting with defective functional responses (MIP-1ß, CD107a) at the same time. Furthermore, NK cell activation was negatively correlated with patient HBsAg levels while NK function correlated with patient age. CONCLUSIONS: The differential regulation of NK cell phenotype and function suggests that activation of NK cells in patients with low serum HBsAg levels may contribute to HBV clearance.

Intestinal tolerance requires gut homing and expansion of FoxP3+ regulatory T cells in the lamina propria.

Tolerance to food antigen manifests in the absence and/or suppression of antigen-specific immune responses locally in the gut but also systemically, a phenomenon known as oral tolerance. Oral tolerance is thought to originate in the gut-draining lymph nodes, which support the generation of FoxP3(+) regulatory T (Treg) cells. Here we use several mouse models to show that Treg cells, after their generation in lymph nodes, need to home to the gut to undergo local expansion to install oral tolerance. Proliferation of Treg cells in the intestine and production of interleukin-10 by gut-resident macrophages was blunted in mice deficient in the chemokine (C-X3-C motif) receptor 1 (CX3CR1). We propose a model of stepwise oral tolerance induction comprising the generation of Treg cells in the gut-draining lymph nodes, followed by migration into the gut and subsequent expansion of Treg cells driven by intestinal macrophages.

Absence of Atg7 in the liver disturbed hepatic regeneration after liver injury.

BACKGROUND AND AIMS: Autophagy is a critical process in cell survival and the maintenance of homeostasis. However, the implementation of therapeutic approaches based on autophagy mechanisms after liver damage is still challenging. METHODS: We used a hepatospecific Atg7-deficient murine model to address this question. RESULTS: We showed that the proliferation and regeneration capacity of Atg7-deficient hepatocytes was impaired. On the one hand, Atg7-deficient hepatocytes showed steady-state hyperproliferation. On the other hand, external triggers such as partial hepatectomy (PHx) or cell transplantation did not induce hepatocellular proliferation or liver repopulation. After PHx, hepatocyte proliferation was strongly decreased, accompanied by high mortality. This increase in mortality could be overcome by pharmacological mTOR inhibition. In accordance with hepatocyte hypoproliferation after damage, Atg7-deficient hepatocytes failed to repopulate the liver in a hepatic injury model. Atg7-deficient mice showed hepatic hypertrophy, transient cellular hypertrophy, and high transaminase levels followed by strong perisinusoidal/pericellular fibrosis with age. Their elevated modified hepatic activity index (mHAI) was almost exclusively due to apoptosis without any inflammation. These parameters were associated with variations in the triglyceride content and compromised lipid droplet formation after PHx. Mechanistically, we also observed a modulation of HGF, PAK4, NOTCH3 and YES1, which are proteins involved in cell cycle regulation. CONCLUSION: We demonstrated the important role of autophagy in the regeneration capacity of hepatocytes. We showed the causative relationship between autophagy and triglycerides that is essential for promoting liver recovery. Finally, pharmacological mTOR inhibition overcame the impact of autophagy deficiency after liver damage and prevented mortality.

Autoimmune hepatitis induction can occur in the liver.

The priming of T cells in the liver is widely accepted. Nonetheless, it is controversial whether immune activation in autoimmune hepatitis (AIH) occurs in the liver or in the spleen. To address this issue, we splenectomized mice and induced experimental murine AIH (emAIH) with an adenovirus (Ad)-expressing formiminotransferase cyclodeaminase (FTCD). Post-splenectomy, the experimental mice developed emAIH to a higher extent than the control mice. In addition, splenectomized mice harboured more intrahepatic B cells and a disproportionately small number of regulatory T cells (Tregs) within a reduced T cell population at the site of inflammation. These results imply that the spleen is not the site of AIH induction. In contrast, the spleen seems to have a protective function since the pathological score was more severe in splenectomized animals. These findings have important implications for the aetiology of AIH.

9-PAHSA Prevents Mitochondrial Dysfunction and Increases the Viability of Steatotic Hepatocytes.

Nonalcoholic fatty liver disease (NAFLD) is quickly becoming the most common liver disease worldwide. Within the NAFLD spectrum, patients with nonalcoholic steatohepatitis (NASH) are at the highest risk of developing cirrhosis and disease progression to hepatocellular carcinoma. To date, therapeutic options for NASH patients have been ineffective, and therefore, new options are urgently needed. Hence, a model system to develop new therapeutic interventions is needed. Here, we introduce two new in vitro models of steatosis induction in HepG2 cells and primary murine hepatocytes. We used a recently discovered novel class of bioactive anti-inflammatory lipids called branched fatty acid esters of hydroxyl fatty acids. Among these bioactive lipids, palmitic-acid-9-hydroxy-stearic-acid (9-PAHSA) is the most promising as a representative nondrug therapy based on dietary supplements or nutritional modifications. In this study, we show a therapeutic effect of 9-PAHSA on lipotoxicity in steatotic primary hepatocytes and HepG2 cells. This could be shown be increased viability and decreased steatosis. Furthermore, we could demonstrate a preventive effect in HepG2 cells. The outcome of 9-PAHSA administration is both preventative and therapeutically effective for hepatocytes with limited damage. In conclusion, bioactive lipids like 9-PAHSA offer new hope for prevention or treatment in patients with fatty liver and steatosis.

Regulatory T cells engineered with a novel insulin-specific chimeric antigen receptor as a candidate immunotherapy for type 1 diabetes.

Adoptive immunotherapy with ex vivo expanded, polyspecific regulatory T cells (Tregs) is a promising treatment for graft-versus-host disease. Animal transplantation models used by us and others have demonstrated that the adoptive transfer of allospecific Tregs offers greater protection from graft rejection than that of polyclonal Tregs. This finding is in contrast to those of autoimmune models, where adoptive transfer of polyspecific Tregs had very limited effects, while antigen-specific Tregs were promising. However, antigen-specific Tregs in autoimmunity cannot be isolated in sufficient numbers. Chimeric antigen receptors (CARs) can modify T cells and redirect their specificity toward needed antigens and are currently clinically used in leukemia patients. A major benefit of CAR technology is its "off-the-shelf" usability in a translational setting in contrast to major histocompatibility complex (MHC)-restricted T cell receptors. We used CAR technology to redirect T cell specificity toward insulin and redirect T effector cells (Teffs) to Tregs by Foxp3 transduction. Our data demonstrate that our converted, insulin-specific CAR Tregs (cTregs) were functional stable, suppressive and long-lived in vivo. This is a proof of concept for both redirection of T cell specificity and conversion of Teffs to cTregs.

Hepatic T Cell Tolerance Induction in An Inflammatory Environment.

For the development of autoimmune hepatitis (AIH), genetic predisposition and environmental triggers are of major importance. Although experimental AIH can be induced in genetically susceptible mice, the low precursor frequency of autoreactive T cells hampers a deeper analysis of liver-specific T cells. Here, we established a system where the model antigen hemagglutinin (HA) is expressed exclusively in hepatocytes of Rosa26-HA mice following administration of a replication deficient adenovirus expressing Cre recombinase (Ad-Cre). Under these conditions, hepatocytes mimic the generation of altered-self neoantigens. To follow autoreactive T cells during AIH, we adoptively transferred HA--specific Cl4-TCR and 6.5-TCR T cells into Ad-Cre infected -Rosa26-HA mice. Alternatively, Rosa26-HA mice have been crossed with TCR transgenic mice that were infected with Ad-Cre to break hepatic tolerance and induce the expression of the HA antigen as a hepatic self-antigen. Surprisingly, neither adoptive transfer nor a very high precursor frequency of autoreactive T cells was able to break tolerance in the context of adenoviral infection. The low proliferation of the antigen experienced autoreactive T cells despite the presence of the autoantigen and inflammation points to anergy as a potential tolerance mechanism. This model underscores the crucial importance of genetic susceptibility to break tolerance against hepatic autoantigens.

The influence of genetic predisposition and autoimmune hepatitis inducing antigens in disease development.

Autoimmune hepatitis (AIH) is defined as a chronic liver inflammation with loss of tolerance against hepatocytes. The etiology and pathophysiology of AIH are still poorly understood because reliable animal models are limited. Therefore, we recently introduced a model of experimental murine AIH by a self-limited adenoviral infection with the AIH type 2 antigen formiminotransferase cyclodeaminase (FTCD). We could demonstrate that break of humoral tolerance towards liver specific autoantigens like FTCD and cytochrome P450 2D6 (CYP2D6) is not dependent on the genetic background. However, the development of AIH in autoantibody positive animals is determined by genetic background genes. We could also show that the break of humoral tolerance is necessary but not sufficient for the development of AIH. In contrast the break of tolerance against the ubiquitously expressed nuclear antigens (ANAs) is strictly dependent on genetic predisposition. Priming with the UGA suppressor tRNA-associated protein (soluble liver antigen; SLA) is a strong inducer of ANA reactivity, but not sufficient to cause AIH development thereby questioning the importance of anti-SLA immune response as an important driver in AIH. Monogenetic mutations such as Aire-deficiency can cause AIH in otherwise genetically resistant strains. CONCLUSION: The results have important implications for our understanding of the pathophysiology of AIH development and for the interpretation of humoral antibody responses in AIH.

Preferential accumulation of T helper cells but not cytotoxic T cells characterizes benign subclinical rejection of human liver allografts.

Subclinical rejection (SCR) is a common event in protocol biopsies after liver transplantation (LT). So far the interpretation of the underlying histological changes and clinical significance is limited. Previous studies were restricted to SCR manifestations within the first weeks after transplantation with limited follow-up. We analyzed clinical data from our prospective protocol biopsy program and found late SCR (at least 3 months after transplantation) to be a common event (41/94 patients). SCR manifested much later than acute cellular rejection (ACR). In the second year after transplantation, the SCR incidence in protocol biopsies reached a plateau of approximately 25% and remained at this level until the latest observed manifestations more than 5 years after transplantation. During a median follow-up of 32 months after SCR, no acute or chronic rejection, relevant graft fibrosis, graft loss, or liver-related death occurred even without specific therapy for SCR. Immunophenotyping of liver biopsies during SCR showed that similar to ACR, the composition of intrahepatic T cells depended on the severity of histological rejection. However, SCR showed a different pattern of infiltrating T cells with a stronger accumulation of CD4(+) cells, an increasing CD4(+) /CD8(+) ratio, and an increasing CD4(+) forkhead box P3 (FOXP3)(+) regulatory T cell (Treg)/CD8(+) ratio, which was not seen in ACR. These intrahepatic T cell patterns were not reflected in the peripheral blood. In conclusion, late SCR after LT has a good clinical prognosis, and it seems safe to leave it untreated. This benign clinical course compared to ACR is associated with intrahepatic T cell infiltration patterns showing less cytotoxic T cells and more CD4(+) FOXP3(+) Tregs. Liver Transplantation 22 943-955 2016 AASLD.

Autoimmune hepatitis in a murine autoimmune polyendocrine syndrome type 1 model is directed against multiple autoantigens.

UNLABELLED: Autoimmune polyendocrine syndrome type 1 (APS-1) is caused by mutations of the autoimmune regulator (AIRE) gene. Mouse studies have shown that this results in defective negative selection of T cells and defective early seeding of peripheral organs with regulatory T cells (Tregs). Aire deficiency in humans and mice manifests as spontaneous autoimmunity against multiple organs, and 20% of patients develop an autoimmune hepatitis (AIH). To study AIH in APS-1, we generated a murine model of human AIH on a BALB/c mouse background, in which Aire is truncated at exon 2. A subgroup of 24% of mice is affected by AIH, characterized by lymphoplasmacytic and periportal hepatic infiltrates, autoantibodies, elevated aminotransferases, and a chronic and progressive course of disease. Disease manifestation was dependent on specific Aire mutations and the genetic background of the mice. Though intrahepatic Treg numbers were increased and hyperproliferative, the intrahepatic CD4/CD8 ratio was decreased. The targets of the adaptive autoimmune response were polyspecific and not focussed on essential autoantigens, as described for other APS-1-related autoimmune diseases. The AIH could be treated with prednisolone or adoptive transfer of polyspecific Tregs. CONCLUSION: Development of AIH in APS-1 is dependent on specific Aire mutations and genetic background genes. Autoimmune response is polyspecific and can be controlled by steroids or transfer with Tregs. This might enable new treatment options for patients with AIH.

Isolation of human antigen-specific regulatory T cells with high suppressive function.

Adoptive transfer of regulatory T (Treg) cells could be an alternative to chronic immunosuppression for prevention of allogeneic graft rejection. While polyspecific Treg cells can prevent immune responses under lymphopenic conditions, Ag-specific Treg cells are needed to treat autoimmunity and graft rejection. Yet, reliable markers for Ag-specific Treg cells are missing. We report that latency-associated peptide (LAP) and glycoprotein A repetitions predominant (GARP) can identify human Ag-specific Treg cells. In addition, we show that the depletion of CD154(+) cells from LAP(+) or GARP(+) Treg cells increases the Treg-cell purity to over 90%, as assessed by epigenetic analysis. These Ag-specific Treg cells can be isolated magnetically and might contribute to the development of GMP-based protocols. In addition, Ag-specific Treg cells are functionally far superior to CD4(+) CD25(high) or CD4(+) CD25(high) CD127(low) Treg cells in vitro and in preventing strong alloreactions in humanized mice. They could, therefore, have a high therapeutic potential for the control of alloimmune, autoimmune, and allergic immune responses in patients.

Intrahepatic regulatory T cells in autoimmune hepatitis are associated with treatment response and depleted with current therapies.

BACKGROUND & AIMS: Autoimmune hepatitis (AIH) is a chronic autoimmune liver disease usually requiring life-long immunosuppression. The mechanisms for disease initiation and chronicity are largely unknown. A contribution of deficient regulatory T cells (Tregs) in the blood was controversially discussed recently. So far investigations in the target organ have been limited to single parameter analysis in untreated AIH. METHODS: We retrospectively analysed the pattern of liver infiltrating T, B and regulatory T cells quantitatively with simultaneous multicolour immunofluorescence before (n=45) and under (n=31) therapy in adult AIH type 1 (AIH-1) patients. RESULTS: Intrahepatic CD4(+) cells dominate over CD8(+) at diagnosis, but with increasing disease activity the CD4(+)/CD8(+) ratio approached one. While there is no change of Tregs in the blood, they are enriched with effector T cells (Teffs) within the liver of patients with untreated AIH-1 with a constant Treg/Teff ratio. Even more importantly, immunosuppression mostly with steroids and azathioprine caused a disproportional loss of intrahepatic Tregs. Patients reaching biochemical remission had higher intrahepatic Treg/Teff and Treg/B cell ratios compared to patients failing to reach remission. In vitro proliferation of Tregs seemed to be more suppressed by prednisolone than expansion of Teffs. Furthermore, intraportal B cells correlated with serum IgG suggesting an autochthonous intrahepatic IgG production. CONCLUSIONS: Intrahepatic Tregs are rather enriched than numerically deficient in untreated AIH-1. The disproportional decrease of intrahepatic Tregs during therapy might explain high relapse rates after discontinuation of immunosuppression. Thus, future therapies increasing intrahepatic immunoregulation might be better suited for long-term control of AIH.

Genetic predisposition and environmental danger signals initiate chronic autoimmune hepatitis driven by CD4+ T cells.

UNLABELLED: Autoimmune hepatitis (AIH) is defined as a chronic liver disease with loss of tolerance against liver tissue eventually leading to cirrhosis if left untreated. 80%-90% of patients can be treated with a life-long immunosuppression. Unfortunately, there are strong drug-related side effects and steroid-refractory patients. Therefore, there is a need for a model system to investigate the complex immunopathogenesis of this chronic disease and subsequently to develop new therapeutic interventions. We developed a new model of experimental murine AIH (emAIH) by a self-limited adenoviral infection with the hepatic autoantigen formiminotransferase cyclodeaminase (FTCD). After an initial transient hepatitis there was a chronic evolving AIH, finally leading to portal and lobular fibrosis. We could show that the genetic predisposition provided by the NOD background was essential for creating a fertile field for the development of liver-specific autoimmunity. However, a strong environmental trigger was additionally necessary to initiate the disease. Besides the break of humoral tolerance, T-cell tolerance against hepatic self-antigens was also broken and CD4(+) T cells were identified as essential drivers of the disease. As the disease was successfully treated with prednisolone and budesonide, the model will be helpful to develop and test new therapeutic interventions. CONCLUSION: We developed a new murine AIH model closely resembling AIH in patients that explains the mechanisms of AIH pathophysiology. In addition, emAIH provides options to test therapeutic alternatives for patients not achieving remission, with reduced side effects of chronic nonspecific immunosuppression.

The importance of liver microcirculation in promoting autoimmune hepatitis via maintaining an inflammatory cytokine milieu--a mathematical model study.

In autoimmune diseases, inflammatory cytokine concentrations are important for initiating and maintaining the status of autoimmunity. Autoimmune hepatitis (AIH) is an inflammatory liver disease characterized by a loss of immune tolerance against specific antigens located in hepatocytes. During the progression of the disease, antigen-presenting cells and different classes of T-helper cells secrete specific cytokines important for maintaining the disease. As these cytokines are secreted into the local liver environment, the blood flow in liver sinusoids might influence the local cytokine concentration. Considering the liver tissue as a porous medium, based on Darcy׳s law, the microcirculation within a liver lobule was modelled. Using realistic physiological pressure differences and tissue permeabilities, the blood velocity inside the sinusoids could be calculated and validated with blood velocity data obtained via Orthogonal Polarization Spectral Imaging (OPSI). Furthermore, oxygen consumption is modelled to obtain Rappaport׳s acinus model. Finally, steady state spatial distributions of secreted cytokines within the liver lobule could be estimated for specified realistic production rates of T-helper cells. It could be demonstrated that the characteristics of the liver microcirculation have an important impact on establishing inflammatory cytokine levels within the portal fields and the vascular septa promoting the occurrence of interface hepatitis.

Tipping the balance in autoimmunity: are regulatory t cells the cause, the cure, or both?

Regulatory T cells (Tregs) are a specialized subgroup of T-cell lymphocytes that is crucial for maintaining immune homeostasis and preventing excessive immune responses. Depending on their differentiation route, Tregs can be subdivided into thymically derived Tregs (tTregs) and peripherally induced Tregs (pTregs), which originate from conventional T cells after extrathymic differentiation at peripheral sites. Although the regulatory attributes of tTregs and pTregs partially overlap, their modes of action, protein expression profiles, and functional stability exhibit specific characteristics unique to each subset. Over the last few years, our knowledge of Treg differentiation, maturation, plasticity, and correlations between their phenotypes and functions has increased. Genetic and functional studies in patients with numeric and functional Treg deficiencies have contributed to our mechanistic understanding of immune dysregulation and autoimmune pathologies. This review provides an overview of our current knowledge of Treg biology, discusses monogenetic Treg pathologies and explores the role of Tregs in various other autoimmune disorders. Additionally, we discuss novel approaches that explore Tregs as targets or agents of innovative treatment options.

PD-L1 blockade effectively restores strong graft-versus-leukemia effects without graft-versus-host disease after delayed adoptive transfer of T-cell receptor gene-engineered allogeneic CD8+ T cells.

Adoptive transfer (AT) of T cells forced to express tumor-reactive T-cell receptor (TCR) genes is an attractive strategy to direct autologous T-cell immunity against tumor-associated antigens. However, clinical effectiveness has been hampered by limited in vivo persistence. We investigated whether the use of major histocompatibility complex-mismatched T cells would prolong the in vivo persistence of tumor-reactive TCR gene expressing T cells by continuous antigen-driven proliferation via the endogenous potentially alloreactive receptor. Donor-derived CD8(+) T cells engineered to express a TCR against a leukemia-associated antigen mediated strong graft-versus-leukemia (GVL) effects with reduced graft-versus-host disease (GVHD) severity when given early after transplantation. AT later after transplantation resulted in a complete loss of GVL. Loss of function was associated with reduced expansion of TCR-transduced T cells as assessed by CDR3 spectratyping analysis and PD-1 up-regulation on T cells in leukemia-bearing recipients. PD-L1 blockade in allogeneic transplant recipients largely restored the GVL efficacy without triggering GVHD, whereas no significant antileukemia effects of PD-L1 blockade were observed in syngeneic controls. These data suggest a clinical approach in which the AT of gene-modified allogeneic T cells early after transplantation can provide a potent GVL effect without GVHD, whereas later AT is effective only with concurrent PD-L1 blockade.

Development of Beta-Amyloid-Specific CAR-Tregs for the Treatment of Alzheimer's Disease.

BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative disease that remains uncured. Its pathogenesis is characterized by the formation of ß-amyloid (Aß) plaques. The use of antigen-specific regulatory T cells (Tregs) through adoptive transfer has shown promise for the treatment of many inflammatory diseases, although the effectiveness of polyspecific Tregs is limited. Obtaining a sufficient number of antigen-specific Tregs from patients remains challenging. AIMS AND METHODS: To address this problem, we used an antibody-like single-chain variable fragment from a phage library and subsequently generated a chimeric antigen receptor (CAR) targeting ß-amyloid. RESULTS: The ß-amyloid-specific CARs obtained were stimulated by both recombinant and membrane-bound Aß isolated from the murine brain. The generated CAR-Tregs showed a normal Treg phenotype, were antigen-specific activatable, and had suppressive capacity. CONCLUSION: This study highlights the potential of CAR technology to generate antigen-specific Tregs and presents novel approaches for developing functional CARs.